Courtship and Mating in Phausis Reticulata (Coleoptera: Lampyridae): Male Flight Behaviors, Female Glow Displays, and Male Attraction to Light Traps

1290 Florida Entomologist 97(4) December 2014

COURTSHIP AND MATING IN PHAUSIS RETICULATA (COLEOPTERA: LAMPYRIDAE): MALE FLIGHT BEHAVIORS, FEMALE GLOW DISPLAYS, AND MALE ATTRACTION TO LIGHT TRAPS

1* 2 3 Raphaël De Cock , Lynn Faust and Sara Lewis 1Evolutionary Ecology Group, University of Antwerp, B-2610 Antwerp, Belgium

211828 Couch Mill Road, Knoxville TN 37932 USA

3Department of Biology, Tufts University, Medford MA 02155 USA

*Corresponding author; E-mail: [email protected]

Supplementary material for this article in Florida Entomologist 97(4) (December 2014), including a video of P. reticulata mating behavior, is online at http://purl.fcla.edu/fcla/entomologist/browse

Abstract

In contrast to most other North American fireflies that use flash dialogs for courtship, males of Phausis reticulata Say (Coleoptera: Lampyridae) (Fender 1966), often called blue ghost fireflies, glow as they fly slowly over the forest floor searching for flightless, neotenic females that are likewise bioluminescent. Recently, these Blue Ghost firefly displays have become increasingly popular as ecotourist attractions. Nevertheless, surprisingly little work has been done on P. reticulata courtship and mating behavior, and little is known of female oviposition patterns. Extensive field observations were conducted at 2 locations in Tennessee USA, leading to the description of new categories of male mate-searching search behaviors and nightly display activities. Spectrophotometric measures of bioluminescence were similar in

both sexes (λmax = 552 nm). There was a 3-fold variation in female body size, and size was correlated to the number of dorsal photic organs (3 to 9). Field experiments suggested that, in addition to their courtship glows, P. reticulata females might also emit a sex pheromone. Males were more likely to approach artificial lures that had a greater number of female-like light spots. Mean copulation duration was 7.9 ± SE 1.3 min and females oviposited an average clutch size of 31 eggs (n = 3; range 27-37). Females guard their eggs and this is described for the first time in any lampyrid. We present photographs of eggs and first instar larvae. These results indicate that the courtship signaling and mating biology of P. reticulata is more complex than previously thought, and we suggest future research.

Key Words: reproductive ecology, display activity, bioluminescence, sex pheromones, oviposition, larva

Resumen

En contraste con la mayoría de otras luciérnagas norteamericanas que usan diálogos de flash (destellos) para el cortejo, los machos de la luciérnaga fantasma azul,Phausis reticulata Say (Fender 1966) (Coleoptera: Lampyridae), brillan mientras que vuelan lentamente sobre el piso del bosque en busca de hembras no voladoras y neotenicas, que son igualmente bioluminiscentes. Recientemente, estas exhibiciones de la luciérnaga fantasma azul se han vuelto cada vez más populares como atracciones ecoturísticas. Sin embargo, muy poco se ha trabajado en el cortejo y el comportamiento del apareamiento de la Ph. reticulata, y se sabe poco del patrón de oviposición de las hembras. Varios autores han sugerido que las feromonas podrían jugar un papel en el cortejo, pero no se han realizado estudios experi- mentales hasta ahora. El objetivo de este estudio fue proveer información adicional sobre la ecología reproductiva de esta especie de luciérnaga enigmática y bastante atípica. Este estudio provee varios nuevos conocimientos sobre el comportamiento del cortejo y de la eco- logía reproductiva de la luciérnaga fantasma azul norteamericana, Ph. reticulata. En base a las observaciones extensas de campo en 2 sitios en Tennessee, EE.UU., describimos varias nuevas categorías del comportamiento de búsqueda en los machos, la actividad noctura de exhibición de los machos y proveemos las primeras mediciones espectrofotométricas de las señales de cortejo bioluminiscentes emitidas por cada sexo (λmax = 552 nm). Se describe una variación de 3 veces en el tamaño del cuerpo de las hembras, que parece estar asociada con las variaciones en el número de puntos luminosos de las hembras (que van de 3 a 9 puntos de luz) y sus patrones. Los resultados de los experimentos de campo que evalúan las señales De Cock et al.: Courtship and Mating in the Blue Ghost Firefly 1291

del cortejo de las hembras sugieren la posibilidad de que, además de sus brillos de cortejo, las hembras Ph. reticulata también podrían utilizar las feromonas como una señal de cortejo. Además, nuestros experimentos usando señuelos brillantes como los de las hembras sugieren que los machos se acercan preferentemente a los señuelos con más puntos de luz. Por último, proveemos observaciones detalladas de la cópula (el promedio de la duración de la cópula: 7.9 ± 1.3 min SE) y el comportamiento de oviposición de la hembra (el promedio del tamaño del grupo de huevos: 31; n = 3, rango 27-37), que incluye la primera descripción de la hembra de cualquier especie de luciérnaga que vigila los huevos. También fotografiamos los huevos y por primera vez las larvas de primer estadio. Estos resultados indican que la señalización del cortejo y la biología del apareamiento de la Ph. reticulata es mucho más enigmática de lo que se pensaba anteriormente y sugieren nuevos caminos para futuras investigaciones.

Palabras Clave: ecología reproductiva, actividad de exhibición, bioluminiscencia, feromonas sexuales, oviposición, larva

Male Phausis reticulata Say (Coleoptera: Surprisingly little work has been done on P. Lampyridae) (Fender 1966) glow as they fly reticulata courtship and mating behavior. Frick- slowly over the forest floor searching for females Ruppert & Rosen (2008) described courtship be- (Frick-Ruppert & Rosen 2008). In contrast to haviors of both sexes and observed a few success- most other North American fireflies that use ful matings. While some authors have suggested precisely-timed flash dialogs for courtship, both that pheromones might play a role in P. reticulata sexes of P. reticulata emit long-lasting, near con- courtship (Lloyd 1965, 1997a, 1997b, 2004; Frick- tinuous glows. In forested regions of the southern Ruppert & Rosen 2008), no relevant experimental Appalachian Mountains, large numbers of males studies have been conducted. In addition, little is create an eerie glowing display, which gave rise known about female oviposition patterns (but see to the widely used nickname of “blue ghost fire- Wing 1988). fly”. Watching these blue ghost fireflies during the The goal of this study was to provide additional dual-peak flight seasons, i.e., Apr-May for “early insight into the reproductive ecology of this enig- season” and Jun-Jul for “late season (Faust & matic and rather atypical firefly species. Work- Weston 2009), has become increasingly popular, ing with field populations in Tennessee, USA, we with annual tours held at conservationist Don observed P. reticulata courtship and copulation Lewis’s “Firefly Forest” in South Carolina (http:// behaviors, as well as female oviposition behavior. upstateforever.org/portfolios/firefly-forest/) andWe also performed controlled field experiments DuPont State Forest in North Carolina (http:// to investigate courtship signals used by P. reticu- www.dupontforest.com) and other sites attract- lata females, including the relative importance ing ecotourists (http://www.yoursmokies.com/ of pheromones and bioluminescent glows. Addi- firefliesinthesmokies.html). tionally, we report here for the first time spectral Previous work described P. reticulata morphol- measurements of P. reticulata bioluminescent sig- ogy (Fender 1966), geographic distribution (Lloyd nals, intraspecific variation in female light organ 2004; Frick-Ruppert & Rosen 2008), and seasonal patterns, and detailed descriptions of copulation and nightly activity patterns (Frick-Ruppert & and oviposition, including female egg guarding. Rosen 2008). Phausis reticulata exhibits strong sexual dimorphism: while males have normal Materials and Methods wings and can fly, the neotenic females lack both elytra and wings (Lloyd 1971, 1983, 1997a, 1997b, Distribution, Seasonality and Courtship Activity 2004; Cicero 1988; Branham & Wenzel 2003). Be- cause of the specific geographic range and the fe- This study is based on fieldwork conducted male’s larviform appearance, the species has also during 4-19 Jun 2011 and 15-26 Jun 2013 on P. been referred to as the “Appalachian glow-worm” reticulata populations within the Great Smoky (Lloyd 1971; Branham & Wenzel 2003). These Mountains National Park (GSMNP). Co-author flightless females have several spot-like light or- Lynn Faust made additional observations over 22 gans whose glow is visible dorsally through the years (1991-2013) in the GSMNP in Sevier and transparent cuticle (Lloyd 1965; Frick-Ruppert Blount counties and additionally in Morgan, Jef- & Rosen 2008). Although such “glow-worm” fe- ferson and Knox counties, Tennessee, USA. The males are common in Europe, flightless neotenic geographic distribution of P. reticulata is cen- females are rare among North American fireflies. tered in the mountains and valleys of southern Another unusual feature of P. reticulata biology Appalachia, although small populations have is that their males produce light, while the males been reported as far west as Texas and Oklaho- of most other glow-worm fireflies are not lumines- ma (Fender 1966; Lloyd 2004; Frick-Ruppert & cent (De Cock 2009). Rosen 2008). This species typically displays in 1292 Florida Entomologist 97(4) December 2014 moist forested habitats, often with small streams exposed on top of the leaf litter or up to 20 cm on or wetlands nearby, and soils overlain by a thick low vegetation, stumps or fallen branches (Lynn layer of leaf litter (Frick-Ruppert & Rosen 2008; Faust, pers. obs.). We photographed each display- Faust & Weston 2009; Faust 2010). The GSMNP ing female (n = 14) with a Sony Cybershot DSC populations that we studied occurred in Appala- T20 to determine the number, size, and distribu- chian secondary and old-growth oak-hickory for- tion of light organ spots; spot patterns were later est (475-950 m asl), where they often occur sym- transferred to schematized drawing of a P. reticu- patrically with the synchronous firefly, Photinus lata female. These light spots and their position carolinus (Green) (Faust & Weston 2009). We and within the female body can only be determined others have seen P. reticulata displays at some in live glowing females (Frick-Ruppert & Rosen high elevation sites within GSMNP (1280-2025 2008). It was sometimes difficult to determine m), where they inhabit open areas with grasses light spot position exactly, as some were located and sedges bounded by dominant trees of red at the borders between body segments, and inter- spruce, yellow birch, and rhododendron (Frick- nal organs shift their relation to the exoskeleton Ruppert & Rosen 2008; A. Mayor, personal com- during movement. We also estimated female body munication; A. Bedinger, personal communica- length, which we categorized as small (≤ 6 mm), tion). Some lowland populations of P. reticulata medium-large (between 6 and 8 mm) or large (> can also be found on dry forested hilltops and 8 mm). In 2013, we were able to take photomicro- woods and open field margins (LF, personal ob- graphs that enabled us to measure body size more servation), as well as dry habitats on Chilhowee precisely as the area of each female’s pronotum Mountain in the Appalachian foothills (A. C. Cole (head covering). We examined whether the num- Collection, Univ. Tennessee-Knoxville). ber of light spots on females covaried with their Within GSMNP, we focused our studies on the body size using Pearson’s correlation test. P. reticulata mating peak that occurs between We conducted our behavioral observations in mid-Jun and mid-Jul (Lloyd 1965; Frick-Ruppert the laboratory and field using blue-filtered head- & Rosen 2008). This is the second of 2 seasonal lamps, as some but not all other firefly species peaks that have been observed in both lowland show low sensitivity to blue wavelengths (Buck (Knoxville area) and GSMNP P. reticulata popu- 1937; Lall & Worthy 2000). Additional behavioral lations (Faust & Weston 2009): an early-season, observations in the lab were video recorded un- high-density peak occurs in April-May, followed der infrared illumination using a Sony Nightshot by a lower density peak that occurs Jun-Jul. It video camera. is not currently known whether these constitute a single species or distinct subspecies. Displays Field Observations of Courtship Behaviors and Tests of begin when P. reticulata males start flying and Courtship Signals with Live Females glowing in full darkness at 20:30 h (~40 min after sunset), and courtship activity has ceased by In Jun 2011, we observed P. reticulata court- midnight. Male flight activity depends on ambi- ship behavior in the field by placing glowing fe- ent light levels (fewer males display at full moon), males into an area where courting males flew in and is curtailed by heavy rainfall or very dry con- high density. Three females (2 large and 1 small, ditions. see Table 1) were individually placed in 9 cm di- Field observations were made nightly from am petri dishes furnished with moist paper and 20:00-24:00 by patrolling ~4 km tracks through P. native leaf litter for shelter. Each petri dish was reticulata habitat at several locations in GSMNP: sealed with fine-mesh fabric to prevent males on the west side of Burnt Mountain (at 730 m el- from reaching and copulating with the females, evation), near Park Headquarters (475 m), on the and each was placed on a 1 m² square of dark north side of Sugarlands Mountain (845 m), and fabric to facilitate behavioral observations once on the NW flank of Mount LeConte (946 m). Civil males had landed. twilight was between 21:15-21:23 pm EDT, and In Jun 2013, we conducted a field experiment full moon fell on 15 Jun 2011 and 23 Jun 2013. Air to investigate the courtship signals, including temperatures during male flight periods ranged possible pheromones, emitted by P. reticulata fe- between 16-25 °C. Additional observations on P. males. We compared the number of attracted to reticulata reproductive behavior were made by live field-collected females assigned to 3 treat- co-author Lynn Faust during the early and late ments: emitting Glow-only (n = 5), emitting Glow peaks in Knoxville and the Cumberland moun- + possible Pheromone (n = 6) and emitting pos- tains, and are also reported here. sible Pheromone-only (n = 7). In each treatment The flightless, tinyP. reticulata females are females were placed individually into cylindri- difficult to find (Frick-Ruppert & Rosen 2008), cal cardboard containers (6 cm high, 9 cm diam) but we located glowing females by searching after with moist paper for humidity and local leaf litter sunset in shaded areas with thick, moist leaf litter. for shelter. For the Glow-only treatment (Glow), Females were often found nestled into the leaf lit- each container was sealed with an airtight trans- ter, although some displaying females were found parent cover that allowed males to see the fe- De Cock et al.: Courtship and Mating in the Blue Ghost Firefly 1293

Table 1. Details on Phausis reticulata females found in 2011 and 2013; initial date found, date of death or release (25.vi.13), light spot number, body length and size class, oviposition date and clutch size. (/ = inapplicable or no data).

Date # Light Oviposition Clutch Date/time found of death spots Length – size class date size 04.VI.11 >23:00 14.VI.11 4 6mm - small / / 04.VI.11 >23:00 13.VI.11 7 8mm - medium-large 11.VI.2011 infertile (?) clump 06.VI.11 22.30 14.VI.11 3 5mm - small / / 06.VI.11 21:30 18.VI.11 7 8mm - medium-large 17.VI.2011 37# 07.VI.11 ~22:00 15.VI.11 7 9mm - large 11.VI.2011 infertile (?) clump 09.VI.11 ~22:00 16.VI.11 6 10mm - large / / 10.V.13 >22:00* 25.V.11 4 7mm - medium 18.V.2011 27# 18.VI.13 >22:00 Released 4 small / / 18.VI.13 >22:00 Released 7 medium-large / / 18.VI.13 >22:00 Released 9 large / / 19.VI.13 >23:20 Released 8 very large / / 21.VI.13 23:15 Released 6 medium-large / / 21.VI.13 23:15 Released 7 large / / 21.VI.13 21:39** Released with eggs 9 medium-large 24.VI.2013 30# 22.VI.13 >23:20 Released 7 large / /

*“Early season, Knoxville low-land” female, mating when collected, eggs hatch 24-25 VI.13: 4 larvae died 26.VI.11. **Mating when collected, remated 21.VI.13 and 23.VI.13. #Showed egg-guarding behavior.

male’s glow. For the Pheromone-only treatment the number of P. reticulata males in the following (Pheromone), each container was covered with categories: mesh that allowed any possible sex pheromones 1) airspace males, defined as any male that to disperse yet prevented males from contacting passed through the 1 m radius around each fe- females; these containers were also fitted with male, 2) influenced males, defined as any male cardboard shields that prevented males from see- that altered their flight behavior near females, in- ing the female’s glow. For the glow + pheromone cluding direct approaches, sudden turns towards treatment (Glow + Pheromone), containers were or within the 1 m radius, circling around the fe- only covered with mesh, which allowed males to male’s container, and spotlight behavior (defined see the female’s glow and allowed any possible sex below), 3) males that landed on the ground within pheromones to disperse. Females were acclima- 1 m radius of the female’s container, and 4) males tized to their containers for at least 12 h before that landed on the female’s container. We also re- the start of experiment. Because female avail- corded the sequence of behaviors shown by males, ability was limited (National Park Service regu- recording which airspace males landed within lations strongly limit collection within GSMNP), 1m, and whether they subsequently landed on females were re-assigned to different treatments the container. We compared the number of males across several nights. observed within the airspace of females from the Tests were conducted over 4 nights. Each 3 experimental treatments and early (< 22:10) vs. night, the females were set out in their natural late (>22:10) in the flight period using two-way habitat ~20-30 min before the male flight period ANOVA. began, with each container centered on a 30 × 30 We considered a female to have successfully at- cm dark fabric to facilitate counts of any males tracted a male if any males landed within 1 m or that landed near the container (a minimum 2 m on her container. We compared females’ success distance between containers was used). Based on in attracting males (yes/no) between the 3 treat- previous observations of male approach behaviors ments using a Freeman-Halton exact contingency in 2011, we also marked out an area 1 m in radius table test. For females in the Glow and Glow + around each container that we defined as the “air- Pheromone treatments, we also tested whether a space” from which a flying male would be able to female’s attractiveness (measured as the propor- see a glowing female. tion of airspace males that landed) was related to Each female’s container was observed continu- how many light spots she showed using Pearson’s ously from the beginning of the male flight period correlation test. at 21:30 until male flight activity ended. Every 10 During these experiments, we also estimated min during this observation period, we counted male flight activity by periodically counting the 1294 Florida Entomologist 97(4) December 2014 number of males flying (and glowing) every 10 www.agcchem.com), which makes surfaces too min. We used these data to divide the number slippery for insects to climb. We also added ~1 cm of males observed at each 10 min interval by the of water to the bottom of each trap. To complete maximum number counted in any interval on each trap, a green Betalight glow lure was sus- that particular night and location to yield male pended on a thread just under the opening of the activity at each time point as a proportion. In funnel. this way, we were able to account for variations We used these traps to test 2 specific hypothe- in male density between nights and locations to ses about what characteristics of female glow sig- estimate temporal patterns of male activity over nals are attractive to P. reticulata males. The first the entire flight period. hypothesis we tested was whether males were more strongly attracted to lures with a greater Testing Male Attraction to Glow Signals using Artificial number of glow spots. We compared the number Lures of males attracted to traps containing lures with 4-spots versus 8-spots. We added a realistic fe- We conducted field experiments using artifi- male body outline by applying a 3 × 10 mm piece cial light lures and traps to investigate whether of whitish masking tape surrounding the spots. males would be differentially attracted to differ- These tests were performed on 22 and 23 Jun ent female glow patterns of female light organs. 2013 with 12 replicates per treatment. Using such glow lure-trap combinations provides The second hypothesis we tested was whether an effective method to study which characteris- males need the pale body outline of females to tics of female glow signals are attractive to males identify an appropriate mate, in addition to the (Schwalb 1961). Lure glow patterns can easily be glow spots. To do this, we compared the number modified to test natural variants of the female of males attracted to traps containing 8-spotted signal (e.g. changing number and patterns of lures that had a pale, female-like body outline light spots etc.), or to test male preferences for created with masking tape as above vs. control different glow colors. When mounted in an appro- 8-spotted lures with dark body outline. Tests were priate trap design that collects males, the number performed on 24 and 25 Jun 2013 at Elkmont in of trapped males can be used as an estimate for GSMNP with 12 replicates per treatment. Lure the attractiveness of the glow lure. data were analyzed using exact binomial tests Glow lures consisted of reusable lures using and Fisher’s exact tests on 2 × 2 contingency Betalights (SRB Technologies, Pembroke Ontar- tables. io: www.betalight.com), glass tubes in which tri- tium activates a pigment that provides a constant Measuring Induced Glow Behavior and Bioluminescent dim glow closely resembling the light emitted by Emission Spectrum a P. reticulata female. Betalight lures have many advantages. They glow for decades, do not require Many lampyrid larvae and adults glow when electricity or batteries, and are weatherproof. We they are disturbed, which is thought to serve as made preliminary tests of Betalights in 2 differ- an aposematic signal (Sivinski 1981; Underwood ent colors, yellow (λmax = 574 nm) and green (λmax et al. 1997; De Cock & Matthysen 1999, 2003; De = 530 nm) but employed the green for further ex- Cock 2009). We investigated this aspect of P. retic- periments. ulata biology in laboratory studies conducted dur- Each glow lure was made by inserting a 2 × 25 ing Jun 2011 and 2013. Field-collected adults (10 mm cylindrical Betalight into a 40 mm-long sec- males and 14 females) were separated by sex and tion cut from an opaque black plastic straw that kept in small containers provided with moist pa- had been pierced with several small holes. We per and local leaf litter. To test induced glowing, positioned these holes so that the light shining the fireflies were left covered and undisturbed in a through the straw from the glowing Betalight in- dark room for 10-15 min. We then presented them side created a pattern that closely resembled the with stimuli designed to mimic a sequence of in- light spot pattern seen in P. reticulata females. creasing threat intensity: nearby noise (rustling These lures were mounted inside funnel bottle of plastic bag), substrate vibration (knocking on traps made by cutting off the neck of a 2 liter so- table), air currents (opening containers and blow- da bottle and inverting the upper tapering section ing on them), touching their bodies gently (with onto the base of the bottle, effectively forming a a feather), and gently grasping their bodies (with funnel trap (Ineichen 2004; Ineichen & Rüttiman soft forceps). We also made note of any induced 2012). Phausis reticulata seem to avoid landing glowing behavior that we observed in the field or on shiny surfaces, so we painted both the interior as a result of handling in the lab. and exterior of the funnels matte dark green. To We measured the bioluminescent emission prevent males from flying out of our funnel traps, spectrum of both sexes using a RedTide USB 650 we coated the interior of the trap and funnel with spectrophotometer with a QP600-2-VIS-BX optic Fluon®, an aqueous dispersion of polytetrafluoro- probe (Ocean Optics, Dunedin, Florida USA). P. ethylene (AGC Chemicals, Exton, Pennsylvania: reticulata males light organs are located on the De Cock et al.: Courtship and Mating in the Blue Ghost Firefly 1295 fifth and sixth ventral sternites; these appear checked every 5 min to determine when copula- as a whitish-yellow, kidney-shaped area occupy- tion occurred. ing nearly the entire segment (drawing in Lloyd To check whether females would remate, a fe- 1965; Frick-Ruppert & Rosen 2008). Males can male that we found mating in the field on 23 Jun control their luminescence by glowing from one 2013 was placed in a container with a different or both light segments (Frick-Ruppert & Rosen wild-caught male. We also recorded mating and 2008). egg-laying behaviors exhibited by early-season Working in a darkened room, males were im- females from Knoxville (early Jun 2011 and May mobilized by holding them ventral side up on 2013). Lastly, a late-season P. reticulata female cotton batting then covering each male with fine from GSMNP was mated in captivity with a mesh. This disturbance generally induced glows Knoxville male. lasting several s, and the optic probe could be held Some of the data on female glow activity dur- directly against the glowing light organ. We ob- ing glow and pheromone experiments in the field, tained replicate scans with high signal strength as well as colored versions of the Figs. 2, 3, 4, 8 and low noise for 2 males using an integration and 9 can be seen online in Florida Entomologist time of 2 sec and boxcar width of 10: 3 scans (each 97(3) (September 2014) at http://purl.fcla.edu/ over 700 counts) were obtained from one male and fcla/entomologist/browse. 2 scans (each over 600 counts) were obtained from the other male. We combined these by standardiz- Results ing each scan to minimum intensity = 0 and maximum = 1 to obtain an average bioluminescence Field Observations of Courtship Behaviors spectral curve for P. reticulata males. We used similar methods to measure the bio- Male Search Behaviors luminescence spectrum of 2 P. reticulata females. During the nightly display period when females In the late-season P. reticulata populations were glowing, we took measurements by holding that we studied in GSMNP, estimated peak male the optic probe directly against the female’s abdo- densities were ~10 males per 100 m². Males began men. However, because the female glow is quite flying and glowing in search of females at 30 min faint and the light spots are quite small, it was after sunset, near the end of civil twilight (21:30 difficult to properly position the 600 µm probe EDT, Fig. 1). Male display activity quickly rose to opening over the glow spot(s). To obtain readings a maximum by 21:30-21:50, after which activity we needed to use longer integration times, and declined sharply. We saw a small but noticeable there was considerable noise in the measure- secondary rise in male display activity near 23:00 ments. We obtained replicate scans (each over (Fig. 1). After 23:10, only a few males were dis- 700 counts) from 2 females: 4 scans were obtained playing, and almost none were seen flying by mid- from one female using an integration time of 10 night. At the beginning of their flight period, P. re- sec and boxcar width of 10, and 2 scans were ob- ticulata males flew only a few centimeters above tained from another female using an integration the forest floor, rising to fly 20-50 cm above the time of 20 sec and boxcar width of 10. Again, we ground later at night. Males typically flew slowly combined these female scans by standardizing in meandering paths, but sometimes males flew each (to minimum intensity = 0 and maximum = higher (50 cm-1m) and more quickly (10 cm per 1) to obtain an average bioluminescence spectral sec). curve for P. reticulata females. Based on our observations of hundreds of male search flights, we described the following catego- Mating and Oviposition Behaviors ries of male search behavior: Patrol flight – groups of 2-5 males travel in parallel flight paths, mov- Because so little is known concerning the ing a single direction (often through open areas). mating behavior of P. reticulata fireflies, we con- Zooming – males fly high and fast in a straight ducted observations in the lab on mating inter- path. Late-flying males (after 22:00) often showed actions and investigated the possibility of female this behavior when flying along open roads or remating. Mating observations were initially per- paths. Spotlighting – when males fly low (< 20 formed in darkness, but we found that males and cm above the substrate), their glowing light or- females would also readily mate under daylight gan casts a greenish circle onto the ground. These conditions. In Jun 2013, 7 P. reticulata females eerie dim lights are most apparent to human ob- were each placed in a petri dish with wild-caught servers when males fly over level roads or paths. males. Behavioral interactions were video-re- Low-flying males may use this reflected light for corded, and copulation durations measured with altitude control, or to avoid crashing into under- a stopwatch. Mating observations were made on story vegetation. Jitterbugging – males hover low large vs. small P. reticulata females (2 each) in over a single spot and fly within a single horizon- Jun 2011; females were acclimatized for 30 min tal plane using small, jerky movements. After in a dark room before males were introduced. We jitterbugging, males sometimes land and then 1296 Florida Entomologist 97(4) December 2014

Fig. 1. Nightly flight activity ofPhausis reticulata males, measured at 10 min intervals by counting glows from flying males. Counts were standardized to maximum interval count observed at each night and location, then aver- aged (n = 7, bars show mean + 1 SE). take flight again. This behavior was often ob- s in close vicinity. Some males eventually ap- served near our experimental females, and may proached quite closely, and then dropped direct- allow males to locate partially hidden females by ly onto or within 2 cm of the glowing source. In changing their visual perspective. Elevator flight the course of our study we had opportunities to – males fly up and down in a vertical plane, cov- observe males’ reactions to many different types ering up to 60 cm. We often saw groups of 2 to 5 of glow sources and males appeared more likely males exhibiting this behavior. Again, this may to inspect and land on lures that more closely allow males to locate females by changing their resembled P. reticulata females (e.g. female- perspective. Hopping – males fly in short (< 20 sized pattern of several light spots). These ob- cm) hops along the ground or through vegetation, servations suggested that males could not detect repeatedly landing and taking flight again. Males female-like glows beyond 1 m distance. On one were often observed hopping in dense vegetation occasion we noticed that males would only ap- or as they approached a glowing female. Magnet proach and inspect but not land at a female-like flight – males fly directly toward a female from far glow pattern made with phosphorescent paint. away (3 to 6 m). We observed this behavior among However once the glow lure was partly covered late-flying males in response to experimental fe- with some leaves, 4 males landed directly on the males. This could represent male response to a light source within 2 min. These approaches and pheromonal signal released by females, as female landings only occurred during peak male activity glows do not appear to be detected by males at (21:45-22:00); later males seemed to ignore this these distances. glow lure. We observed P. reticulata males in the field We observed many instances of spider preda- that were clearly attracted to dimly glowing tion on P. reticulata males by orb weavers, sheet light sources, such as small dots of phospho- web weavers and even a black widow (Latrodec- rescent paint, and small Betalights, but not to tus mactans Fabricius 1775). On one occasion, 5 stronger light sources, such as LEDs or 9V light- males were trapped in a single web. During one bulbs. As males flew over, they would often circle night of peak display in the early season Knox back to inspect these fainter light sources. Males County population (7th May 2013), 13 of 143 P. flew around these glows for up to 15 sec at ~ 20 reticulata censused by their glows turned out to cm height, moving in small circles or spirals ~10 be males caught in spider webs. Glows emitted to 80 cm in diameter, often while jitterbugging by trapped males seemed to attract flying males, and spotlighting, often spending up to 10 or 15 which sometimes also become ensnared. De Cock et al.: Courtship and Mating in the Blue Ghost Firefly 1297

Female Display Behavior Tests of Courtship Signals with Live Females

Phausis reticulata females displayed by glow- We conducted a field experiment to investi- ing from on top of leaf litter or soil, and some gate the courtship signals used by P. reticulata were found up to 20 cm on low vegetation. Most females by comparing how many males were at- females commenced their display near the be- tracted to live P. reticulata females assigned to 3 ginning of the males’ flight period, though some treatments: emitting Glow-only (n = 5), emitting females began later. When they failed to mate, Glow + possible Pheromone (n = 6) and emitting females continued glowing after peak male flight possible Pheromone-only (n = 7). In this experi- activity. Females that had already mated in the ment, the number of males we observed flying lab left their shelters after sunset, climbing to a through the 1 m radius airspace around each fe- fixed position and assumed the typical display male on a given night (Fig. 6a) did not differ sig- glow posture on subsequent nights, suggesting nificantly among treatments (two-way ANOVA, that females will mate more than once. Based treatment F(2, 32) = 1.92, P = 0.164). There was a on our observations of the same experimental marginally significant decline in the number of females over several nights, it appears that fe- airspace males during the late (> 22:10 h) vs. ear- males initially began displaying around the time ly (< 22:10 h) flight period (time (1,32)F = 4.00, P = of peak male activity or later, but on subsequent 0.054), which was concordant with declining male nights these unmated females often began dis- activity during the late flight period. There was playing even before males had started flying. At no significant treatment * time interaction (F(2, 32) one field site, we found a glowing female (with = 1.50, P = 0.239). For this experiment, we consid- 7 light spots) positioned at the sloped edge of a ered a female to be successful in attracting a male forest path; although she appeared highly vis- passing through her airspace if he subsequently ible to us, she failed to attract any males even landed either directly on or within a 1 m radius of though she displayed continuously from 22:00 her container (Fig. 6b). The proportion of airspace until 23:15 when we left the site. males that landed was highest for females in the Female body size was highly variable, and Glow + Pheromone treatment, followed by Glow we found a 3-fold difference in size measured as females and then Pheromone females. Glow and pronotal area (Fig. 2a, b). Larger females often Glow + Pheromone treatments show about simi- showed highly distended abdomens that were lar proportions if early and late airspace males filled with white or yellowish eggs clearly visible are combined, while the Pheromone treatment through the dorsal cuticle (Fig. 2c). clearly shows lowest proportions in such a con- Previous studies have reported 4 to 6 light sideration. spots in P. reticulata females (Lloyd 1965; Frick- Across all treatments, female success in- Ruppert & Rosen 2008), but our study found this creased over the flight period. As indicated by the number to range between 3 and 9 spots (Fig. 3), wide error bars in Fig. 6b, we saw considerable and these were visible both dorsally and ven- variation among females within treatments. For trally. We monitored spot patterns for females example, the most successful female was a large kept in captivity, and these maintained the same individual with 7 light spots (Glow treatment): light spot pattern throughout our study. However, she attracted 28 males into her airspace, and 20 light spot position was highly variable among fe- of these males subsequently landed within 1 m of males (Fig. 4); only 3 of 14 females showed fully her, and 16 of these (thus 57% of airspace males) symmetrical arrangements (2 with 4 spots, one eventually landed on her container. In another with 6 spots; Fig. 4a, f), while the remainder were trial she attracted 1 male that landed directly on asymmetrical. Such asymmetry manifests either her container. Yet other brightly glowing females as different numbers of spots on each side or as in the Glow treatment only had a single airspace differences in light spot positions within a body male landing within 1m, and no other females at- segment. tracted males directly to their containers. Female light spot pattern varied with body Although relatively few males landed near fe- size. The smallest females usually showed a males in the Pheromone (no glow visible) treat- pattern of 4 light spots (e.g. female shown in ment, we observed 3 males that had been flying as Fig. 4a, b, c): 2 spots were located on abdomi- high as 1 m above the female drop directly down nal segment 1, and 2 spots located on either onto these females’ containers, and 3 additional segment 6 or 7. The general pattern in larger males landed within the 1m perimeter. Additional females was a pair of spots in the first 2 ab- males were observed approaching these Phero- dominal segments, with more (usually smaller) mone females from up to 3 m away, some showing spots positioned in other abdominal segments magnet flight and others approaching by jitter- (e.g. females shown in Figs. 3 and 4). In 2013 bugging and eventually landing nearby. we measured female body size as pronotal area, We also considered the attraction success for and found this was positively correlated with each test female, i.e. whether any males landed the number of light spots (Fig. 5). near her on a given night. We observed that 57% 1298 Florida Entomologist 97(4) December 2014

Fig. 2. Size variation among Phausis reticulata females (a, b). Female with eggs visible through the transparent dorsal cuticle (c). (Photos a, b: R. De Cock; photo c: L. Faust). A colored version of this figure can be seen online in Florida Entomologist 97(4) (December 2014) at http://purl.fcla.edu/fcla/entomologist/browse . of females in the 7 Pheromone trials successfully fully attracted males after 22:10, thus after peak attracted males, compared to 60% of those in the 5 male activity. In the Pheromone treatment, 2 out Glow trials, and 100% of females in Glow + Phero- of 7 females attracted males during peak male mone treatment. However, these proportions were activity (before 22:10), and 2 other females were not significantly different across the 3 treatments successful after 22:10, thus after peak male activ- (Freeman-Halton exact test of 2 × 3 contingency, ity. two-tailed P = 0.2). Additionally, we noticed only We tested whether a female’s attractiveness 2 out of 5 females in the Glow treatment attracted (measured as the proportion of airspace males any males after 22:10, whereas 5 out of 6 females that landed) was related to how many light spots in the Glow + Pheromone treatment still success- she showed, using only data from treatments De Cock et al.: Courtship and Mating in the Blue Ghost Firefly 1299

Fig. 3. Variation among Phausis reticulata females in the number, size and position of light spots used in courtship displays. (Photos: R. De Cock.). A colored version of this figure can be seen online in Florida Entomologist 97(4) (December 2014) at http://purl.fcla.edu/fcla/entomologist/browse .

with visible glow signals (thus Glow and Glow + this was not significant (Fisher’s exact test of 2 × Pheromone). We found only a weak correlation 2 contingency table, P = 0.2). between the number of female light spots and her The second hypothesis we tested using this attractiveness (n = 10, Pearson r = 0.39, P = 0.3) method was whether the pale body color of P. re- that explained about 15% of the variation. ticulata females, in addition to their glow, might During many hours of observation, we made help males to locate mates. Males hovering above additional notes on male flight behaviors outside females and spotlighting might be able to use the and inside the female perimeter which give ex- reflection from the whitish body to distinguish fe- tra anecdotal clues about the possible presence of males from other glowing objects, such as other female pheromones compared to the importance males trapped in spider webs, fungus gnats or of female glows (supplied in Supplementary Re- other firefly species. We found that 13 males were sults). attracted to and captured in traps baited with pale-colored 8-spot lures resembling females, Testing Male Attraction to Glow Signals Using Glow while 11 males were caught with dark-colored Lures 8-spot lures (n = 12 traps each). Treating each of the 24 males captured as an independent trial, The first hypothesis we tested using the glow males were not preferentially attracted to the lure trap method was whether P. reticulata males pale lures (exact binomial test of no preference, would be more strongly attracted to lures with a P = 0.27). Similarly, we found no significant dif- greater number of glow spots. A total of 20 males ference in capture success between pale-colored were attracted to and captured in traps baited lures (75%) and dark-colored lures (34%: Fisher’s with an 8-spot lure, while only 5 males were cap- exact test of 2 × 2 contingency table, P = 0.2). tured with 4-spot lures. Treating each of these 25 males captured as an independent trial, males Bioluminescent Emission Spectrum appear to be preferentially attracted to 8-spot rather than 4-spot lures (binomial test of equal Although flyingP. reticulata males appear to probability that a male will enter either trap type, emit a bluish glow when viewed from a distance P = 0.002). However, the assumption of indepen- or from above (hence the name “blue ghosts”), dent trials may be violated if the glows emitted when viewed directly and close-by, the light from by trapped males attracted additional males into their lantern appears yellowish-green. When traps. Thus, we re-analyzed these data consider- viewed from certain angles, flying males give the ing only whether or not each trap had captured impression of emitting a slightly modulated flick- any males (rather than the number of males). ering glow. When flying males are viewed from This more conservative approach showed that 8 below however, they emit a steady glow. out of 12 8-spot lures captured males (66% cap- Spectrophotometer measurements confirm ture success) compared to 4 out of 12 4-spot lures the lime-green color of male P. reticulata biolu- (33% capture success); although the difference in minescence (Fig. 7), as the emission spectrum capture success was in the predicted direction, shows a maximum at 552 nm. The 50% spectral 1300 Florida Entomologist 97(4) December 2014

Fig. 5. The relationship between female body size measured as pronotal area and the number of glow spots observed in Phausis reticulata females in Jun 2013. Female body size, and number of light spots show a positive Pearson correlation (n = 10, Pearson r = 0.78, P = 0.007).

tral curve) spans 154 nm and lies between 500 nm (turquoise green) and 654 nm (bright poppy red). Although females were difficult to measure due to their much weaker glows and tiny light spots, the overall shape of the female spectrum closely resembles that of males (Fig. 7). The max- imal wavelength of female bioluminescence lies at 547 nm, close to the male maximum. The 50% spectral bandwidth spans 63 nm and lies between 523 nm and 586 nm, which closely matches that of males. The 10% spectral bandwidth spans 237 nm and lies between 497 and 736 nm, although female spectra were quite noisy at wavelengths above 620 nm.

Induced Glow Behavior

When disturbed or threatened, in 20% of cases (50 trials: 10 males tested 5 times) P. reticulata males exhibited induced glow behavior. When lightly touched most of the responsive males lit both photic organs, though others glowed only from one of both. The response did not seem to Fig. 4. Schematics showing variation and asym- be shown consistently within the same individual metry in light spot patterns among 14 “late season” fe- but happened more randomly. When presented male Phausis reticulata observed in Jun 2011 and Jun with threatening stimuli, males often feigned 2013from the GSMNP. Female body margins visible death (thanatosis) by becoming immobile and through transparent broader tergites are indicated by depressing their antennae. One male showed a dotted line. Small type females 5-6mm (a- c) bear 3 thanatosis at first touch, but glowed in reaction to 4 light organs, while large type females 8-12mm (d- n) showed 6 up to 9 lanterns. A colored version of this to subsequent touch. figure can be seen online in Florida Entomologist 97(4) Induced glow in response to disturbance (December 2014) at http://purl.fcla.edu/fcla/entomolo- was observed in 59% of 14 P. reticulata females gist/browse . tested: 31% of the females glowed in response to substrate vibration and 28% glowed only when touched. These induced glow responses varied bandwidth spans 61 nm and lies between 525 nm between differently sized females. Compared (blue-green) and 586 nm (yellow-orange). The to small females, large females responded more 10% spectral bandwidth (or the base of the spec- frequently (41% vs. 18% of 22 trials), responded De Cock et al.: Courtship and Mating in the Blue Ghost Firefly 1301

Fig. 7. Bioluminescence spectra for adult Phausis reticulata.

Mating and Oviposition Behavioral Observations

We observed P. reticulata mating behavior both in the wild and in the laboratory. When placed together with a female, males typically approached, antennated, and climbed onto the female’s back. When multiple males were placed together with a single female, males sometimes competed by butting into one another with their pronotum and flipping other males away. Females seemed to be able to exert some choice over their mating part- ners by tucking under their terminal segments and moving away from certain males. Intromis- sion began in the dorsal mount position (Fig. 8), Fig. 6. a. Number of Phausis reticulata males that flew through the airspace (within 1 m radius) of ex- which lasted ~ 1 min before the male rotated 180 perimental females assigned to 3 treatments (mean + 1 degrees to continue copulation in the tail-to-tail SE); PHEROM (n = 7; female emitting possible phero- position (see video in Florida Entomologist 97(4) mone, no glow), GLOW (n = 5; female emitting glow, no (December 2014) online at http://purl.fcla.edu/ pheromone), and GLOW+PHEROM (n = 6; female emit- fcla/entomologist/browse ). Copulation durations ting glow and possible pheromone). Counts are shown ranged from 5 min 30 s to 10 min, with a mean divided between early flight period (before 22:10) and (± 1 SE) duration of 7.9 ± 1.3 min (n = 5 pairs). late flight period (after 22:10). b. Proportion of these Males did not glow during copulation, and under airspace males that landed directly on or within 1 m natural conditions female glows were gradually of females (mean + 1 SE) in the same 3 treatments and time periods. extinguished. Copulations ended subtly; as the male disengaged, he often remained near the female for several min. Two females that had already mated in the wild were observed to remate more quickly (<1 sec vs. 4-5 sec), and had longer with new males in the lab. glow durations (5 – 30 sec vs. < 5 sec.). Yet when In 13 mating trials conducted in captivity, 7 fe- they were disturbed repeatedly, the smaller females were ignored by males even after they con- males responded more quickly and with longer tacted and antennated the female. This occurred glow durations. Large females glowed more fre- in all trials with small females that had 4 or less quently in response to substrate vibrations: this glow spots. In these trials the females kept crawl- included one ovipositing female and one female ing and males showed no particular interest in that was guarding eggs. Both large and small them, even up to 47 min after introduction. Also females glowed in response to touch (14%). Fe- during a trial with a large female and 2 males, males that were glowing gradually stopped when the female kept crawling slowly but elicited no they were disturbed by bright light or threatening response from either male even after antennal touch. We did not observe any females turning on contact. None of the females glowed during mat- their glow in response to males flying overhead, a ing experiments conducted in captivity. For com- behavior reported by Lloyd (1965). parison, Frick-Ruppert & Rosen (2008) observed 1302 Florida Entomologist 97(4) December 2014

Fig. 8. Pair of Phausis reticulata fireflies in the initial dorsal mount phase of copulation (male positioned above female). (Photo: R. De Cock). A colored version of this figure can be seen online in Florida Entomologist 97(4) (De- cember 2014) at http://purl.fcla.edu/fcla/entomologist/browse .

3 matings under fully natural conditions of which and kept at natural temperature and photoperi- 2 were successful. Yet, they also report 2 success- od, 30% of one clutch that contained 27 eggs took ful matings in 3 trials when males were placed 34-35 days to hatch. Newly hatched first instar near dark females during manipulated mating larvae were unpigmented and ~1.5 mm in length experiments in the field. (Fig. 9c). Mated P. reticulata females laid their eggs sin- gly and in groups (Fig. 9a) over a period of several Discussion hours, with a mean clutch size of 31 eggs (n = 3; range 27 to 37 eggs). Freshly laid eggs were oval, Courtship Signals and Behaviors: Glows and Phero- yellow-tan in color, and about 0.7 mm long. After mones ovipositing, females (n = 4) curled their bodies tightly around their eggs (Fig. 9b), using one or Despite its common name of blue ghost firefly, more legs to grasp them. Once they had oviposit- our spectrophotometric measurements demon- ed, females rejected any additional advances from strate that bioluminescence of both sexes is yel- males and continued their egg-guarding behav- low-green, peaking at around 552 nm. This glow ior. When we gently disturbed these females, they color closely resembles that reported for other briefly abandoned their clutch to move a short glow-worm fireflies: 550 –547 nm inL. noctiluca distance away; they then returned and resumed (Sala-Newby et al. 1996; De Cock 2004), 550 nm a position tightly clutching their eggs. After being in Lamprohiza splendidula (Schwalb 1961) and threatened once daily, one 2013 female returned Asian species Pyrocoelia miyako (refs. see: Day to grasp her clutch 6 days in a row, until the day of 2009). Glow-worm fireflies, which are character- her death. When eggs were misted every few days ized by flightless, neotenic females that emit con- De Cock et al.: Courtship and Mating in the Blue Ghost Firefly 1303

Fig. 9. Phausis reticulata oviposition, egg-guarding behavior, and newly hatched larva: a) Freshly laid softer, slightly oval eggs; b) round, more rigid eggs ready to eclose; c) and e) females guarding eggs 2012, 2011; d) Newly eclosed larva. (Photos a, d, e: R. De Cock; photos b, c: L. Faust). A colored version of this figure can be seen online in Florida Entomologist 97(4) (December 2014) at http://purl.fcla.edu/fcla/entomologist/browse . 1304 Florida Entomologist 97(4) December 2014 tinuous light to attract flying males, are unusual gested for other glow-worm fireflies by Yiu 2013). in North America (Lloyd 1997b; Branham & Wen- Another hypothesis, not mutually exclusive, is zel 2003). Surprisingly, the yellow to amber biolu- that such glows allow the males to illuminate minescence colors typically seen in many flashing (Lloyd 1968), and thus avoid, potential hazards. species (Pyractomena spp., Photinus spp., Photu- Our observations and glow lure experiments indi- ris spp. (Day 2009; Lall et al. 2009) and Luciolinae cate that male P. reticulata are attracted to other (Day 2009) have not been reported from any glow- glows, including those produced by dying fireflies worm firefly species. Potential advantages of this captured by predators (e.g. spiders, harvestmen, wavelength may be that lime-green glows of the or Photuris females; Lewis et al. 2012), fireflies sedentary females is best enhanced by reflection trapped in puddles, or the glows of Orphelia fulto- off the (equally green) surrounding vegetation, ni fungus gnats. By illuminating the vicinity with and that glow-worm fireflies start activity well their glows before landing, males may be able to after sunset and thus avoid signal-to-noise issues distinguish the pale-bodied outlines surrounding from vegetated surroundings (Lall et al. 2009). the glows of conspecific females from glows asso- Yiu (2013) distinguished 2 signaling systems ciated with other hazards. Thirdly, these flying used by glow-worm fireflies: either both sexes are males might glow to signal that they are apose- luminescent or males are entirely non-lumines- matically defended to nocturnal flying predators, cent or are only faintly luminous (usually from such as bats or caprimulgids (Moosman et al. larval lanterns and only glowing on disturbance). 2009), or even to Photuris fireflies that are spe- In P. reticulata both sexes are luminous with cialized firefly predators themselves but seem to the males having specialized adult light organs. reject P. reticulata males (Lewis et al. 2012). This characteristic is shared with Lamprohiza As noted in previous studies (Lloyd 1964, splendidula (Linné, 1767) from Central Europe Frick-Ruppert & Rosen 2008), P. reticulata often (Schwalb 1961; De Cock 2009) and several Asian occurs in forested habitats with dense understory Diaphanes and Pyrocoelia species (Chen 2003; vegetation. In such obstacle-filled habitats, sig- Ohba 2004; Yiu 2012, 2013; Wong 2013), South naling systems may evolve that use a combina- American Cratomorphus spp. and Magnocu- tion of pheromones for long-range attraction and lus spp. from Brazil (Viviani 2012). P. reticulata bioluminescence for better short-range localiza- closely resembles European L. splendidula shar- tion (Lewis & Cratsley 2008). Among fireflies, ing similar habitats, behaviors, habitats and ecol- direct evidence for volatile pheromones in sexual ogy. Notably, females of both species show an un- communication has thus far been restricted to usual arrangement of light organs for lampyrids diurnally active species (De Cock & Matthysen (Lloyd 1997b; Branham & Wenzel 2003): small 2005; Lloyd 1972, 1997b; Ohba 2004). To date no glow spots spread laterally over the abdomen and lampyrid pheromones have been isolated or char- visible from both ventral and dorsal sides. This acterized (Lewis 2009) except for some species of glow spot arrangement resembles that of phen- the Asian firefly genusPyrocoelia (Shibue et al. godid glow-worms, Rhagophtalmidae or larval 2000). It had previously been hypothesized that luminescence in click beetles (Sivinski 1981). P. reticulata might use pheromones (Lloyd 1965; Similar to Lamprohiza species (De Cock 2009), it but see Lloyd 1997b). Both our study and Frick- is possible that the glow spots of P. reticulata fe- Ruppert & Rosen (2008) observed males flying males are inherited from the larval stages. A lack directly upwind toward females. of typical adult light organs with reflectors may Our study represents the first attempt to ex- explain why Phausis females are noticeably dim perimentally unravel the relative importance of compared to their own males and also to females female pheromones and glow signals in the court- of other similar glow-worm firefly species. ship system of P. reticulata fireflies. The obser- The function of male glows remains unknown. vations and results from our courtship signal It has been suggested (Lloyd 1965, 1997, Yiu experiment provide suggestive, though inconclu- 2013) that male light production might induce sive, evidence that female-produced pheromones females to glow. Although Lloyd (1971) reported may play a role in P. reticulata courtship. With observing P. reticulata females beginning to glow and without putative pheromone release, glow- in response to males’ glows overhead, such female ing females did not show statistically significant glow responses to males were never observed in differences in their attractiveness measured both our study or by Frick-Ruppert & Rosen (2008). as the number of airspace males and the propor- Instead, in our experiments females began glow- tion of these males that landed. It is possible that ing independently within min of one another on the cardboard cover used to block the female vi- multiple nights. Based on our observations we sual signal in our experimental design may have propose 3 alternative hypotheses for male glows somehow interfered with the production (e.g. if in P. reticulata. First, male glows might trigger fe- pheromone release is triggered by females see- males to produce a second courtship signal, such ing male glows) or dissemination of any female- as a sex pheromone (see below) that allows males produced pheromones. We also cannot rule out to identify them as appropriate mates (also sug- that our experiment might have been confounded De Cock et al.: Courtship and Mating in the Blue Ghost Firefly 1305 by the presence of wild females nearby, with the may result from fluctuating asymmetry and thus possibility that high female densities might have another honest advertisement of female fitness overloaded the air with female pheromones. possibly with implications for male mate choice. On the other hand, preliminary evidence Wing (1988) also noted that the asymmetrical ar- comes from observations that males often ap- rangement of light organs on P. reticulata females proached females assigned to the pheromone may even be sufficient to identify individuals. treatment (no glow visible) from downwind and Interestingly, such variation in female light exhibited flight patterns typical of males track- spot number was absent in a lowland population ing a pheromone plume. In addition, we observed of P. reticulata females (Knoxville area), where all 3 males that landed directly on the containers of females show 4 light spots irrespective of body size females in this treatment, while another 3 males (pers. obs. Lynn Faust). It is possible that what is landed within a 1m radius of the female. It seems currently described as P. reticulata actually rep- very unlikely that these landings and approach resents a complex of several species or subspecies, behaviors happened by chance. though this remains to be investigated. Our field observations suggest that the P. reticulata signaling system may be more complex Phausis reticulata Mating Systems than previously thought. At the outset of the flight period, males may rely primarily on light Our results indicate that P. reticulata females signals to locate females, perhaps later switching are polyandrous: not only did mated females con- to pheromones when the pheromone plumes of tinue to emit courtship glow signals on successive relatively fewer unmated females would be more nights, but the females we observed mating in the distinct. Alternatively, unmated females might field readily remated in captivity. Wing (1988) also also release pheromone only later at night. Such reported a P. reticulata female that mated twice possibilities are supported by our experimen- in captivity. This is in contrast to the monogamy tal observations during the late flight period in displayed by the flightless, neotenic females of which Pheromone + Glow females often rapidly another North American firefly, Photinus col- attracted several males, whereas the Glow-only lustrans (Wing 1984, 1988). However, females in females seemed less successful after male peak the Asian glow-worm fireflyPyrocoelia pectoralis activity. Thus, it is possible that P. reticulata were also found to be highly polyandrous, mating courtship signaling strategies may vary both in with up to 7 different males (Fu et al. 2012). It re- terms of the relative importance of different fe- mains to be seen whether multiple matings by P. male signals (glows versus pheromones) as well reticulata females results in increased fecundity as in male search patterns (focus on visual versus or egg hatching success, as documented by Fu et chemical signals), depending on temporal, nightly al. (2012) for Pyrocoelia pectoralis. or seasonal changes in relative densities and sex In other fireflies, prolonged copulation dura- ratios. tions are associated with males transferring a complex spermatophore during mating (Wing Female Light-Producing Spots as an Indicator of Body 1988; Lewis et al. 2004). The relatively short Size copulation times we observed in P. reticulata sug- gests these males do not produce spermatophores There was extensive intraspecific variation in (generally < 10 min; see also Wing 1989). Previ- female body size, which was positively correlated ous work by South et al. (2011) reached a similar with differences in how many light spots females conclusion based on male reproductive anatomy. had. Further, our glow lure experiments provide Expanding on Wing’s (1988) photograph by suggestive evidence that males preferentially ap- Sivinski of a female grasping her egg clutch, our proach female-like lures with a greater number study provides evidence for female egg-guarding of light spots. Further studies should reveal if it behavior in P. reticulata. This behavior not only is the overall brightness or glow spot pattern that includes females curling their bodies around their plays a role. Across many insects, female body egg clutch initially, but continuing to guard their size is correlated with fecundity (Honěk 1993), egg mass until their death (maximally observed and this has also been shown for a firefly (Wing up to 9 days after oviposition). Though abandoning 1989). Thus, our results provide novel evidence the clutch if threatened, females would repeatedly that P. reticulata males, could use female court- return to this guarding position when conditions ship glows as an honest signal of female reproduc- were safe. Females, but not males, glowed to sub- tive potential. strate vibrations indicative of a relatively distant Fluctuating asymmetry has been shown to threat. It seems likely that this behavior evolved be an indicator of fitness (Tomkins & Kotiaho as a way to reduce egg predation most likely act- 2001; Reusch & Muhlhauser 1998; but: Lens et ing as a defensive startling or aposematic signal. al. 2002). We observed considerable asymmetry Interestingly, in the Rhagophthalmidae, which in female glow spot patterns (also seen in Euro- is thought to be a sister clade to the Lampyridae pean L. splendidula; pers. obs. R. De Cock) which (Branham & Wenzel 2003), flightlessRhagoph - 1306 Florida Entomologist 97(4) December 2014

thalmus ohbai females similarly curl around their De Cock, R., and Matthysen, E. 1999. Aposematism eggs. They also glow from several lateral photic or- and bioluminescence: Experimental evidence from gans, apparently producing an aposematic warn- glow-worm larvae (Coleoptera: Lampyridae). Evol. ing display (Ohba et al. 1996). Ecol. 13(7/8): 619-639. In conclusion, this study has provided many De Cock, R., and Matthysen, E. 2003. Glow-worm larvae bioluminescence (Coleoptera: Lampyridae) oper- new insights into the courtship signals and mat- ates as an aposematic signal upon toads (Bufo bufo). ing behavior of P. reticulata, a little-studied North Behav. Ecol. 14(1): 103-108. American firefly. In addition, we hope this work De Cock, R., and Matthysen, E. 2005. Sexual com- inspires further research on what appears to be a munication by pheromones in a firefly,Phosphaenus complex mix of courtship signals in this and other hemipterus (Coleoptera: Lampyridae). Anim. Behav. glow-worm fireflies. Particularly useful will be fu- 70(4): 807-818. ture studies to characterize female pheromones, Faust, L. F. 2010. Natural history and flash reper- and to examine how these signals might change toire of the synchronous fireflyPhotinus carolinus with female ages, mating status, and environ- (Coleoptera: Lampyridae) in the Great Smoky Mountains National Park. Florida Entomol. 93(2): mental conditions. 208-217. Faust, L. F., and Weston, P. A. 2009. Degree-day prediction of adult emergence of Photinus caroli- Acknowlegments nus (Coleoptera: Lampyridae). Physiol. Ecol. 38(5): 1505-1512. We thank Becky Nichols and Adriean Mayor of the Fender, K. M. 1966. The genus Phausis in America GSMNP for guidance, help and assistance. Raphael De North of Mexico (Coleoptera: Lampyridae). North- Cock was funded in 2011 by a research grant from the west Sci. 40: 83-95. Research Foundation Flanders (F.W.O. file K2.064.11N) Frick-Ruppert, J., and Rosen, J. J. 2008. Morphol- and thanks Erik Matthysen for support and advice. We ogy and behavior of Phausis reticulata (Blue Ghost thank Patti Edwards, Kathrin Stanger-Hall and Sarah Firefly). J. North Carolina Acad. Sci.: 124(4): 139- Sander for their enthusiastic company, assistance and 147. support during 2011 field trips, Henri Tellegen of Ocean- Fu, X., South, A., and Lewis, S. M. 2012. Sexual di- Optics Inc. for advice on spectrophotometry, Tristan morphism, mating systems, and nuptial gifts in two Mooij (Betalight b.v., The Netherlands) for providing Asian fireflies (Coleoptera: Lampyridae) J. Insect. information and solutions with Betalights, Jennifer Physiol. 58(11): 1485-1492. Frick-Ruppert and 2 anonymous reviewers for valuable Honěk, A. 1993 Intraspecific variation in body size and comments and suggestions on our manuscript. Special fecundity in insects: A general relationship. 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